Inducible promoter which regulates the expression of a peroxidase gene from maize

ABSTRACT

The present invention provides compositions and methods for regulating expression of heterologous nucleotide sequences in a plant. Compositions include a novel nucleotide sequence for an inducible promoter for the gene encoding ZmPOX24. A method for expressing a heterologous nucleotide sequence in a plant using the promoter sequences disclosed herein is provided. The method comprises stabling incorporating into the genome of a plant cell a nucleotide sequence operably linked to the root-preferred promoter of the present invention and regenerating a stably transformed plant that expresses the nucleotide sequence.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication Nos. 60/628,143, filed on Nov. 16, 2004, and 60/629,947,filed on Nov. 22, 2004, which are herein incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,more particularly to regulation of gene expression in plants.

BACKGROUND OF THE INVENTION

Recent advances in plant genetic engineering have enabled theengineering of plants having improved characteristics or traits, such asdisease resistance, insect resistance, herbicide resistance, enhancedstability or shelf-life of the ultimate consumer product obtained fromthe plants and improvement of the nutritional quality of the edibleportions of the plant. Thus, one or more desired genes from a sourcedifferent than the plant, but engineered to impart different or improvedcharacteristics or qualities, can be incorporated into the plant'sgenome. New gene(s) can then be expressed in the plant cell to exhibitthe desired phenotype such as a new trait or characteristic.

The proper regulatory signals must be present and be in the properlocation with respect to the gene in order to obtain expression of thenewly inserted gene in the plant cell. These regulatory signals mayinclude, but are not limited to, a promoter region, a 5′ non-translatedleader sequence and a 3′ transcription termination/polyadenylationsequence.

A promoter is a DNA sequence that directs cellular machinery of a plantto produce RNA from the contiguous coding sequence downstream (3′) ofthe promoter. The promoter region influences the rate, developmentalstage, and cell type in which the RNA transcript of the gene is made.The RNA transcript is processed to produce messenger RNA (mRNA) whichserves as a template for translation of the RNA sequence into the aminoacid sequence of the encoded polypeptide. The 5′ non-translated leadersequence is a region of the mRNA upstream of the protein coding regionthat may play a role in initiation and translation of the mRNA. The 3′transcription termination/polyadenylation signal is a non-translatedregion downstream of the protein coding region that functions in theplant cells to cause termination of the RNA transcript and the additionof polyadenylate nucleotides to the 3′ end of the RNA.

Expression of heterologous DNA sequences in a plant host is dependentupon the presence of an operably linked promoter that is functionalwithin the plant host. The type of promoter sequence chosen is based onwhen and where within the organism expression of the heterologous DNA isdesired. Where expression in specific tissues or organs is desired,tissue-preferred promoters may be used. Where gene expression inresponse to a stimulus is desired, inducible promoters are theregulatory element of choice. In contrast, where continuous expressionis desired throughout the cells of a plant, constitutive promoters areutilized.

An inducible promoter is a promoter that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer, the DNAsequences or genes will not be transcribed, or will be transcribed at alevel lower than in an induced state. The inducer can be a chemicalagent, such as a metabolite, growth regulator, herbicide or phenoliccompound, or a physiological stress directly imposed upon the plant suchas cold, drought, heat, salt, toxins. In the case of fighting plantpests, it is also desirable to have a promoter which is induced by plantpathogens, including plant insect pests, nematodes or disease agentssuch as a bacterium, virus or fungus. Contact with the pathogen willinduce activation of transcription, such that a pathogen-fightingprotein will be produced at a time when it will be effective indefending the plant. A pathogen-induced promoter may also be used todetect contact with a pathogen, for example by expression of adetectable marker, so that the need for application of pesticides can beassessed. A plant cell containing an inducible promoter may be exposedto an inducer by externally applying the inducer to the cell or plantsuch as by spraying, watering, heating, or by exposure to the operativepathogen.

A constitutive promoter is a promoter that directs expression of a genethroughout the various parts of a plant and continuously throughoutplant development. Examples of some constitutive promoters that arewidely used for inducing the expression of heterologous genes intransgenic plants include the nopaline synthase (NOS) gene promoter,from Agrobacterium tumefaciens, (U.S. Pat. No. 5,034,322), thecauliflower mosaic virus (CaMv) 35S and 19S promoters (U.S. Pat. No.5,352,605), those derived from any of the several actin genes, which areknown to be expressed in most cells types (U.S. Pat. No. 6,002,068), andthe ubiquitin promoter, which is a gene product known to accumulate inmany cell types.

Additional regulatory sequences upstream and/or downstream from the corepromoter sequence may be included in expression constructs oftransformation vectors to bring about varying levels of expression ofheterologous nucleotide sequences in a transgenic plant. Geneticallyaltering plants through the use of genetic engineering techniques toproduce plants with useful traits thus requires the availability of avariety of promoters.

In order to maximize the commercial application of transgenic planttechnology, it may be useful to direct the expression of the introducedDNA in a site-specific manner. For example, it may be useful to producetoxic defensive compounds in tissues subject to pathogen attack, but notin tissues that are to be harvested and eaten by consumers. Bysite-directing the synthesis or storage of desirable proteins orcompounds, plants can be manipulated as factories, or productionsystems, for a tremendous variety of compounds with commercial utility.Cell-specific promoters provide the ability to direct the synthesis ofcompounds, spatially and temporally, to highly specialized tissues ororgans, such as roots, leaves, vascular tissues, embryos, seeds, orflowers.

Alternatively, it may be useful to inhibit expression of a native DNAsequence within a plant's tissues to achieve a desired phenotype. Suchinhibition might be accomplished with transformation of the plant tocomprise a tissue-preferred promoter operably linked to an antisensenucleotide sequence, such that expression of the antisense sequenceproduces an RNA transcript that interferes with translation of the mRNAof the native DNA sequence.

Of particular interest are promoters that are induced by the feeding ofinsect pests. Insect pests are a major factor in the loss of the world'sagricultural crops. Insect pest-related crop loss from corn rootwormalone has reached one billion dollars a year. The western corn rootworm(WCRW) is a major insect pest of corn or maize in many regions of theworld. While not as important a pest as the western corn rootworm, thesouthern corn rootworm and northern corn rootworm may also causesignificant economic damage to corn. Damage from corn rootworms mayresult in increased lodging, reduced drought tolerance and ultimately,crop yield reductions. Another one of the leading pests is Ostrinianubilalis, commonly called the European Corn Borer (ECB).

Since the patterns of expression of a chimeric gene (or genes)introduced into a plant are controlled using promoters, there is anongoing interest in the isolation and identification of novel promoterswhich are capable of controlling expression of a chimeric gene or(genes).

SUMMARY OF THE INVENTION

Compositions and methods for regulating gene expression in a plant areprovided. Compositions comprise novel nucleotide sequences for aninducible promoter that initiates transcription in response to feedingby insect pests. More particularly, a transcriptional initiation regionisolated from maize is provided. Further embodiments of the inventioncomprise the nucleotide sequence set forth in SEQ ID NO:1, a fragment ofthe nucleotide sequence set forth in SEQ ID NO:1, and the plant promotersequences deposited with the American Type Culture Collection (ATCC) onNov. 3, 2004 in bacterial hosts as Patent Deposit No. PTA-6276. Theembodiments of the invention further comprise nucleotide sequenceshaving at least 95% sequence identity to the sequence set forth in SEQID NO:1, and which drive insect pest-inducible expression of an operablylinked nucleotide sequence. Also included are functional fragments ofthe sequence set forth as SEQ ID NO:1 which drive insect pest-inducibleexpression of an operably linked nucleotide sequence.

Embodiments of the invention also include DNA constructs comprising apromoter operably linked to a heterologous nucleotide sequence ofinterest wherein said promoter is capable of driving expression of saidnucleotide sequence in a plant cell and said promoter comprises thenucleotide sequences disclosed herein. Embodiments of the inventionfurther provide expression vectors, and plants or plant cells havingstably incorporated into their genomes a DNA construct mentioned above.Additionally, compositions include transgenic seed of such plants.

Method embodiments comprise a means for selectively expressing anucleotide sequence in a plant, comprising transforming a plant cellwith a DNA construct, and regenerating a transformed plant from saidplant cell, said DNA construct comprising a promoter and a heterologousnucleotide sequence operably linked to said promoter, wherein saidpromoter initiates insect pest-inducible transcription of saidnucleotide sequence in a plant cell. In this manner, the promotersequences are useful for controlling the expression of operably linkedcoding sequences in an inducible manner.

Downstream from and under the transcriptional initiation regulation ofthe promoter will be a sequence of interest that will provide formodification of the phenotype of the plant. Such modification includesmodulating the production of an endogenous product, as to amount,relative distribution, or the like, or production of an exogenousexpression product to provide for a novel function or product in theplant. For example, a heterologous nucleotide sequence that encodes agene product that confers herbicide, salt, cold, drought, pathogen orinsect resistance is encompassed.

In a further embodiment, a method for modulating expression of a gene ina stably transformed plant is provided, comprising the steps of (a)transforming a plant cell with a DNA construct comprising the promoterof the embodiments operably linked to at least one nucleotide sequence;(b) growing the plant cell under plant growing conditions and (c)regenerating a stably transformed plant from the plant cell whereinexpression of the nucleotide sequence alters the phenotype of the plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a northern blot result showing peroxidase expression in roottissue from V5 stage corn plants, both with and without western cornrootworm infestation. No expression was detected in leaves in eithercase. Repot refers to transplanting the plant into fresh soil. Rootdamage caused by this process does not induce peroxidase expression.Thus, this figure demonstrates the pattern of root expression andinducibility by CRW, as predicted by the Lynx MPSS data. Further detailon this blot is provided in Example 2.

FIG. 2 shows the sequence of the Zm-POX24 promoter and its 5′ UTR. Thepositions of the TATA box and other motifs of interest of the promotersequence are indicated.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments of the invention comprise novel nucleotide sequences forplant promoters, particularly an insect-pest inducible promoter for amaize peroxidase gene, more particularly, the Zm-POX24 gene promoter. Inparticular, the embodiments provide for isolated nucleic acid moleculescomprising the nucleotide sequence set forth in SEQ ID NO:1, and theplant promoter sequence deposited in a bacterial host as Patent DepositNo. PTA-6276 on Nov. 3, 2004, and fragments, variants, and complementsthereof.

Plasmids containing the plant promoter nucleotide sequences of theembodiments were deposited on Nov. 3, 2004 with the Patent Depository ofthe American Type Culture Collection (ATCC), at 10801 University Blvd.,Manassas, Va. 20110-2209, and assigned Patent Deposit No. PTA-6276. Thisdeposit will be maintained under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112. The deposit will irrevocablyand without restriction or condition be available to the public uponissuance of a patent. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentaction.

The promoter sequences of the embodiments are useful for expressingoperably linked nucleotide sequences in an inducible manner,particularly in an insect-pest feeding inducible manner. The sequencesalso find use in the construction of expression vectors for subsequenttransformation into plants of interest, as probes for the isolation ofother peroxidase gene promoters, as molecular markers, and the like.

The Zm-POX24 promoter was isolated from maize genomic DNA. The specificmethod used to obtain the Zm-POX24 promoter is described in Example 3 inthe experimental section of this application.

The embodiments encompass isolated or substantially purified nucleicacid compositions. An “isolated” or “purified” nucleic acid molecule, orbiologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. An “isolated” nucleic acid issubstantially free of sequences (including protein encoding sequences)that naturally flank the nucleic acid (i.e., sequences located at the 5′and 3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated nucleic acid molecule can contain less than about 5 kb, 4kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences thatnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived.

Peroxidases are a subclass of oxido-reductases that use a peroxide suchas H₂O₂ as an oxygen acceptor. In plants, peroxidases are monomericproteins whose activities are closely regulated by the plant.Peroxidases function in the synthesis of plant cell walls by promotingthe polymerization of the monolignols coniferyl, ρ-coumaryl, and sinapylalcohol into lignin. Lignification serves to strengthen and reinforceplant cell walls, and increase the stalk strength of the plant. Plantperoxidases are also required for xenobiotic detoxification (reviewed inKorte et al. (2000) Ecotoxicol. Environ. Saf 47:1-26). See alsoWO200270723 and US Patent Application Publication US20030017566, hereinincorporated by reference.

The ZmPOX24 promoter sequence directs expression of operably linkednucleotide sequences in an inducible manner. Therefore, the ZmPOX24promoter sequences find use in the inducible expression of an operablylinked nucleotide sequence of interest. Particularly, the promoter ofthe embodiments acts to induce expression following the feeding activityof an insect pest.

The compositions of the embodiments include isolated nucleic acidmolecules comprising the promoter nucleotide sequence set forth in SEQID NO:1. The term “promoter” is intended to mean a regulatory region ofDNA usually comprising a TATA box capable of directing RNA polymerase IIto initiate RNA synthesis at the appropriate transcription initiationsite for a particular coding sequence. A promoter may additionallycomprise other recognition sequences generally positioned upstream or 5′to the TATA box, referred to as upstream promoter elements, whichinfluence the transcription initiation rate. It is recognized thathaving identified the nucleotide sequences for the promoter regionsdisclosed herein, it is within the state of the art to isolate andidentify further regulatory elements in the 5′ untranslated regionupstream from the particular promoter regions identified herein. Thus,for example, the promoter regions disclosed herein may further compriseupstream regulatory elements such as those responsible for tissue andtemporal expression of the coding sequence, enhancers, and the like. Seeparticularly Australian Patent No. AU-A-77751/94 and U.S. Pat. Nos.5,466,785 and 5,635,618. In the same manner, the promoter elements thatenable inducible expression can be identified, isolated, and used withother core promoters to confer inducible expression. In this aspect ofthe embodiments, a “core promoter” is intended to mean a promoterwithout promoter elements.

In the context of this disclosure, the term “regulatory element” alsorefers to a sequence of DNA, usually, but not always, upstream (5′) tothe coding sequence of a structural gene, which includes sequences whichcontrol the expression of the coding region by providing the recognitionfor RNA polymerase and/or other factors required for transcription tostart at a particular site. An example of a regulatory element thatprovides for the recognition for RNA polymerase or other transcriptionalfactors to ensure initiation at a particular site is a promoter element.A promoter element comprises a core promoter element, responsible forthe initiation of transcription, as well as other regulatory elements(as discussed elsewhere in this application) that modify geneexpression. It is to be understood that nucleotide sequences, locatedwithin introns, or 3′ of the coding region sequence may also contributeto the regulation of expression of a coding region of interest. Examplesof suitable introns include, but are not limited to, the maize IVS6intron, or the maize actin intron. A regulatory element may also includethose elements located downstream (3′) to the site of transcriptioninitiation, or within transcribed regions, or both. In the context ofthe present disclosure, a post-transcriptional regulatory element mayinclude elements that are active following transcription initiation, forexample translational and transcriptional enhancers, translational andtranscriptional repressors, and mRNA stability determinants.

The regulatory elements, or fragments thereof, of the embodiments may beoperatively associated with heterologous regulatory elements orpromoters in order to modulate the activity of the heterologousregulatory element. Such modulation includes enhancing or repressingtranscriptional activity of the heterologous regulatory element,modulating post-transcriptional events, or both enhancing or repressingtranscriptional activity of the heterologous regulatory element andmodulating post-transcriptional events. For example, one or moreregulatory elements, or fragments thereof, of the embodiments may beoperatively associated with constitutive, inducible, or tissue specificpromoters or fragment thereof, to modulate the activity of suchpromoters within desired tissues within plant cells.

The maize ZmPOX24 promoter sequence, when assembled within a DNAconstruct such that the promoter is operably linked to a nucleotidesequence of interest, enables expression of the nucleotide sequence inthe cells of a plant stably transformed with this DNA construct. Theterm “operably linked” is intended to mean that the transcription ortranslation of the heterologous nucleotide sequence is under theinfluence of the promoter sequence. “Operably linked” is also intendedto mean the joining of two nucleotide sequences such that the codingsequence of each DNA fragment remains in the proper reading frame. Inthis manner, the nucleotide sequences for the promoters of theembodiments are provided in DNA constructs along with the nucleotidesequence of interest, typically a heterologous nucleotide sequence, forexpression in the plant of interest. The term “heterologous nucleotidesequence” is intended to mean a sequence that is not naturally operablylinked with the promoter sequence. While this nucleotide sequence isheterologous to the promoter sequence, it may be homologous, or native;or heterologous, or foreign, to the plant host.

It is recognized that the promoters of the embodiments may be used withtheir native coding sequences to increase or decrease expression,thereby resulting in a change in phenotype of the transformed plant.

Modifications of the isolated promoter sequences of the embodiments canprovide for a range of expression of the heterologous nucleotidesequence. Thus, they may be modified to be weak promoters or strongpromoters. Generally, a “weak promoter” is intended to mean a promoterthat drives expression of a coding sequence at a low level. A “lowlevel” of expression is intended to mean expression at levels of about1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000transcripts. Conversely, a strong promoter drives expression of a codingsequence at a high level, or at about 1/10 transcripts to about 1/100transcripts to about 1/1,000 transcripts.

Fragments and variants of the disclosed promoter sequences are alsoencompassed by the embodiments. A “fragment” is intended to mean aportion of the promoter sequence. Fragments of a promoter sequence mayretain biological activity and hence encompass fragments capable ofdriving inducible expression of an operably linked nucleotide sequence.Thus, for example, less than the entire promoter sequence disclosedherein may be utilized to drive expression of an operably linkednucleotide sequence of interest, such as a nucleotide sequence encodinga heterologous protein. Those skilled in the art are able to determinewhether such fragments decrease expression levels or alter the nature ofexpression, i.e., constitutive or inducible expression. Alternatively,fragments of a promoter nucleotide sequence that are useful ashybridization probes, such as described below, may not retain thisregulatory activity. Thus, fragments of a nucleotide sequence may rangefrom at least about 20 nucleotides, about 50 nucleotides, about 100nucleotides, and up to the full-length nucleotide sequences of theembodiments.

Thus, a fragment of a ZmPOX24 promoter nucleotide sequence may encode abiologically active portion of the ZmPOX24 promoter or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of a ZmPOX24promoter can be prepared by isolating a portion of the ZmPOX24 promoternucleotide sequence and assessing the activity of that portion of theZmPOX24 promoter. Nucleic acid molecules that are fragments of apromoter nucleotide sequence comprise at least 15, 20, 25, 30, 35, 40,45, 50, 75, 100, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700,800, 900, or up to the number of nucleotides present in the full-lengthpromoter nucleotide sequence disclosed herein, e.g. 996 nucleotides forSEQ ID NO:1.

The nucleotides of such fragments will usually comprise the TATArecognition sequence of the particular promoter sequence. Such fragmentsmay be obtained by use of restriction enzymes to cleave the naturallyoccurring promoter nucleotide sequence disclosed herein; by synthesizinga nucleotide sequence from the naturally occurring sequence of thepromoter DNA sequence; or may be obtained through the use of PCRtechnology. See particularly, Mullis et al. (1987) Methods Enzymol.155:335-350, and Erlich, ed. (1989) PCR Technology (Stockton Press, NewYork). Variants of these promoter fragments, such as those resultingfrom site-directed mutagenesis and a procedure such as DNA “shuffling”,are also encompassed by the compositions of the embodiments.

An “analogue” of the regulatory elements of the embodiments includes anysubstitution, deletion, or addition to the sequence of a regulatoryelement provided that said analogue maintains at least one regulatoryproperty associated with the activity of the regulatory element of theembodiments. Such properties include directing organ specificity, tissuespecificity, or a combination thereof, or temporal activity, ordevelopmental activity, or a combination thereof.

The term “variants” is intended to mean sequences having substantialsimilarity with a promoter sequence disclosed herein. For nucleotidesequences, naturally occurring variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleotide sequences also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis. Generally, variants of aparticular nucleotide sequence of the embodiments will have at least40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, to 95%,96%, 97%, 98%, 99% or more sequence identity to that particularnucleotide sequence as determined by sequence alignment programsdescribed elsewhere herein using default parameters. Biologically activevariants are also encompassed by the embodiments. Biologically activevariants include, for example, the native promoter sequences of theembodiments having one or more nucleotide substitutions, deletions, orinsertions. Promoter activity may be measured by using techniques suchas Northern blot analysis, reporter activity measurements taken fromtranscriptional fusions, and the like. See, for example, Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.), hereinafter“Sambrook,” herein incorporated by reference. Alternatively, levels of areporter gene such as green fluorescent protein (GFP) or the likeproduced under the control of a promoter fragment or variant can bemeasured. See, for example, U.S. Pat. No. 6,072,050, herein incorporatedby reference.

Methods for mutagenesis and nucleotide sequence alterations are wellknown in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci.USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382;U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein.

Variant promoter nucleotide sequences also encompass sequences derivedfrom a mutagenic and recombinogenic procedure such as DNA shuffling.With such a procedure, one or more different promoter sequences can bemanipulated to create a new promoter possessing the desired properties.In this manner, libraries of recombinant polynucleotides are generatedfrom a population of related sequence polynucleotides comprisingsequence regions that have substantial sequence identity and can behomologously recombined in vitro or in vivo. Strategies for such DNAshuffling are known in the art. See, for example, Stemmer (1994) Proc.Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391;Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J.Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

The nucleotide sequences of the embodiments can be used to isolatecorresponding sequences from other organisms, such as other plants, forexample, other monocots. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequences basedon their sequence homology to the sequence set forth herein. Sequencesisolated based on their sequence identity to the entire ZmPOX24 promotersequence set forth herein or to fragments thereof are encompassed by theembodiments. The promoter regions of the embodiments may be isolatedfrom any plant, including, but not limited to corn (Zea mays), Brassica(Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), sunflower (Helianthus, annuus), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), oats, barley, safflower, vegetables, ornamentals, andconifers.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook, supra. See also Innis et al., eds. (1990)PCRProtocols: A Guide to Methods and Applications (Academic Press, NewYork); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press,New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual(Academic Press, New York). Known methods of PCR include, but are notlimited to, methods using paired primers, nested primers, singlespecific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the ZmPOX24 promotersequence. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook, supra.

For example, the entire ZmPOX24 promoter sequence disclosed herein, orone or more portions thereof, may be used as a probe capable ofspecifically hybridizing to corresponding ZmPOX24 promoter sequences. Toachieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique among ZmPOX24 promotersequences and are generally at least about 10 nucleotides in length,including sequences of at least about 20 nucleotides in length. Suchprobes may be used to amplify corresponding ZmPOX24 promoter sequencesfrom a chosen plant by PCR. This technique may be used to isolateadditional coding sequences from a desired plant or as a diagnosticassay to determine the presence of coding sequences in a plant.Hybridization techniques include hybridization screening of plated DNAlibraries (either plaques or colonies; see, for example, Sambrooksupra).

Hybridization of such sequences may be carried out under stringentconditions. “Stringent conditions” or “stringent hybridizationconditions” are conditions under which a probe will hybridize to itstarget sequence to a detectably greater degree than to other sequences(e.g., at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,including those less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. for at least 30 minutes.Duration of hybridization is generally less than about 24 hours, usuallyabout 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the thermal melting point (T_(m))can be approximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the T_(m) for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the T_(m); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the T_(m); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe T_(m). Using the equation, hybridization and wash compositions, anddesired T_(m), those of ordinary skill will understand that variationsin the stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis preferred to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York), hereinafter “Ausubel”. Seealso Sambrook supra.

Thus, isolated sequences that have inducible promoter activity and whichhybridize under stringent conditions to the ZmPOX24 promoter sequencesdisclosed herein, or to fragments thereof, are encompassed by theembodiments.

In general, sequences that have promoter activity and hybridize to thepromoter sequences disclosed herein will be at least 40% to 50%homologous, about 60%, 70%, 80%, 85%, 90%, 95% to 98% homologous or morewith the disclosed sequences. That is, the sequence similarity ofsequences may range, sharing at least about 40% to 50%, about 60% to70%, and about 80%, 85%, 90%, 95% to 98% sequence similarity.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Preferred, non-limiting examples of such mathematical algorithms are thealgorithm of Myers and Miller (1988) CABIOS 4:11-17; the local homologyalgorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453; the algorithm of Pearson and Lipman (1988) Proc. Natl. Acad.Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc.Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0); the ALIGN PLUS program (Version 3.0,copyright 1997): and GAP, BESTFIT, BLAST, FASTA, and TFASTA in theWisconsin Genetics Software Package of Genetics Computer Group, Version10 (available from Accelrys, 9685 Scranton Road, San Diego, Calif.,92121, USA). The scoring matrix used in Version 10 of the WisconsinGenetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989)Proc. Natl. Acad. Sci. USA 89:10915).

Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN and the ALIGN PLUS programs are based on the algorithm ofMyers and Miller (1988) supra. A PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used with the ALIGNprogram when comparing amino acid sequences. The BLAST programs ofAltschul et al (1990) J. Mol. Biol. 215:403 are based on the algorithmof Karlin and Altschul (1990) supra. BLAST nucleotide searches can beperformed with the BLASTN program, score=100, wordlength=12, to obtainnucleotide sequences homologous to a nucleotide sequence encoding aprotein of the embodiments. BLAST protein searches can be performed withthe BLASTX program, score=50, wordlength=3, to obtain amino acidsequences homologous to a protein or polypeptide of the embodiments. Toobtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST2.0) can be utilized as described in Altschul et al. (1997) NucleicAcids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be usedto perform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See the web site for the National Center for BiotechnologyInformation on the world wide web. Alignment may also be performedmanually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the GAP program with defaultparameters, or any equivalent program. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide or aminoacid residue matches and an identical percent sequence identity whencompared to the corresponding alignment generated by GAP.

The GAP program uses the algorithm of Needleman and Wunsch (1970) supra,to find the alignment of two complete sequences that maximizes thenumber of matches and minimizes the number of gaps. GAP considers allpossible alignments and gap positions and creates the alignment with thelargest number of matched bases and the fewest gaps. It allows for theprovision of a gap creation penalty and a gap extension penalty in unitsof matched bases. GAP must make a profit of gap creation penalty numberof matches for each gap it inserts. If a gap extension penalty greaterthan zero is chosen, GAP must, in addition, make a profit for each gapinserted of the length of the gap times the gap extension penalty.Default gap creation penalty values and gap extension penalty values inVersion 10 of the Wisconsin Genetics Software Package for proteinsequences are 8 and 2, respectively. For nucleotide sequences thedefault gap creation penalty is 50 while the default gap extensionpenalty is 3. The gap creation and gap extension penalties can beexpressed as an integer selected from the group of integers consistingof from 0 to 200. Thus, for example, the gap creation and gap extensionpenalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65 or greater.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, at least 80%, at least 90%, or at least 95%, comparedto a reference sequence using one of the alignment programs describedusing standard parameters. One of skill in the art will recognize thatthese values can be appropriately adjusted to determine correspondingidentity of proteins encoded by two nucleotide sequences by taking intoaccount codon degeneracy, amino acid similarity, reading framepositioning, and the like. Substantial identity of amino acid sequencesfor these purposes normally means sequence identity of at least 60%,70%, 80%, 90%, and at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the T_(m) for the specific sequence at a defined ionic strength andpH. However, stringent conditions encompass temperatures in the range ofabout 1° C. to about 20° C. lower than the T_(m), depending upon thedesired degree of stringency as otherwise qualified herein. Nucleicacids that do not hybridize to each other under stringent conditions arestill substantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

The ZmPOX24 promoter sequence disclosed herein, as well as variants andfragments thereof, are useful for genetic engineering of plants, e.g.for the production of a transformed or transgenic plant, to express aphenotype of interest. As used herein, the terms “transformed plant” and“transgenic plant” refer to a plant that comprises within its genome aheterologous polynucleotide. Generally, the heterologous polynucleotideis stably integrated within the genome of a transgenic or transformedplant such that the polynucleotide is passed on to successivegenerations. The heterologous polynucleotide may be integrated into thegenome alone or as part of a recombinant DNA construct. It is to beunderstood that as used herein the term “transgenic” includes any cell,cell line, callus, tissue, plant part, or plant the genotype of whichhas been altered by the presence of heterologous nucleic acid includingthose transgenics initially so altered as well as those created bysexual crosses or asexual propagation from the initial transgenic. Theterm “transgenic” as used herein does not encompass the alteration ofthe genome (chromosomal or extra-chromosomal) by conventional plantbreeding methods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

A transgenic “event” is produced by transformation of plant cells with aheterologous DNA construct, including a nucleic acid DNA construct thatcomprises a transgene of interest, the regeneration of a population ofplants resulting from the insertion of the transgene into the genome ofthe plant, and selection of a particular plant characterized byinsertion into a particular genome location. An event is characterizedphenotypically by the expression of the transgene. At the genetic level,an event is part of the genetic makeup of a plant. The term “event” alsorefers to progeny produced by a sexual outcross between the transformantand another variety that include the heterologous DNA.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, andprogeny of same. Parts of transgenic plants are to be understood withinthe scope of the embodiments to comprise, for example, plant cells,protoplasts, tissues, callus, embryos as well as flowers, stems, fruits,ovules, leaves, or roots originating in transgenic plants or theirprogeny previously transformed with a DNA molecule of the embodiments,and therefore consisting at least in part of transgenic cells.

As used herein, the term “plant cell” includes, without limitation,seeds suspension cultures, embryos, meristematic regions, callus tissue,leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. The class of plants that can be used in the methodsdisclosed herein is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonousand dicotyledonous plants.

The promoter sequences and methods disclosed herein are useful inregulating expression of any heterologous nucleotide sequence in a hostplant. Thus, the heterologous nucleotide sequence operably linked to thepromoters disclosed herein may be a structural gene encoding a proteinof interest. Genes of interest are reflective of the commercial marketsand interests of those involved in the development of the crop. Cropsand markets of interest change, and as developing nations open up worldmarkets, new crops and technologies will emerge also. In addition, asour understanding of agronomic traits and characteristics such as yieldand heterosis increase, the choice of genes for transformation willchange accordingly. General categories of genes of interest for theembodiments include, for example, those genes involved in information,such as zinc fingers, those involved in communication, such as kinases,and those involved in housekeeping, such as heat shock proteins. Morespecific categories of transgenes, for example, include genes encodingproteins conferring resistance to abiotic stress, such as drought,temperature, salinity, and toxins such as pesticides and herbicides, orto biotic stress, such as attacks by fungi, viruses, bacteria, insects,and nematodes, and development of diseases associated with theseorganisms. Various changes in phenotype are of interest includingmodifying expression of a gene in a plant, altering a plant's pathogenor insect defense mechanism, increasing the plant's tolerance toherbicides, altering plant development to respond to environmentalstress, and the like. The results can be achieved by providingexpression of heterologous or increased expression of endogenousproducts in plants. Alternatively, the results can be achieved byproviding for a reduction of expression of one or more endogenousproducts, particularly enzymes, transporters, or cofactors, or affectingnutrients uptake in the plant. These changes result in a change inphenotype of the transformed plant.

It is recognized that any gene of interest can be operably linked to thepromoter sequences of the embodiments and expressed in a plant.

A DNA construct comprising one of these genes of interest can be usedwith transformation techniques, such as those described below, to createdisease or insect resistance in susceptible plant phenotypes or toenhance disease or insect resistance in resistant plant phenotypes.Accordingly, the embodiments encompass methods that are directed toprotecting plants against fungal pathogens, bacteria, viruses,nematodes, insects, and the like. By “disease resistance” or “insectresistance” is intended that the plants avoid the harmful symptoms thatare the outcome of the plant-pathogen interactions.

Disease resistance and insect resistance genes such as lysozymes,cecropins, maganins, or thionins for antibacterial protection, or thepathogenesis-related (PR) proteins such as glucanases and chitinases foranti-fungal protection, or Bacillus thuringiensis endotoxins, proteaseinhibitors, collagenases, lectins, and glycosidases for controllingnematodes or insects are all examples of useful gene products.

Pathogens of the embodiments include, but are not limited to, viruses orviroids, bacteria, insects, nematodes, fungi, and the like. Virusesinclude tobacco or cucumber mosaic virus, ringspot virus, necrosisvirus, maize dwarf mosaic virus, etc. Nematodes include parasiticnematodes such as root knot, cyst, and lesion nematodes, etc.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer, and the like.Such genes include, for example, Bacillus thuringiensis toxic proteingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756;5,593,881; and Geiser et al. (1986) Gene 48:109); lectins (Van Damme etal. (1994) Plant Mol. Biol. 24:825); and the like.

Genes encoding disease resistance traits include detoxification genes,such as against fumonisin (U.S. Pat. No. 5,792,931) avirulence (avr) anddisease resistance (R) genes (Jones et al. (1994) Science 266:789;Martin et al. (1993) Science 262:1432; Mindrinos et al. (1994) Cell78:1089); and the like.

Herbicide resistance traits may be introduced into plants by genescoding for resistance to herbicides that act to inhibit the action ofacetolactate synthase (ALS), in particular the sulfonylurea-typeherbicides (e.g., the acetolactate synthase (ALS) gene containingmutations leading to such resistance, in particular the S4 and/or Hramutations), genes coding for resistance to herbicides that act toinhibit action of glutamine synthase, such as phosphinothricin or basta(e.g., the bar gene), or other such genes known in the art. The bar geneencodes resistance to the herbicide basta, the nptII gene encodesresistance to the antibiotics kanamycin and geneticin, and the ALS geneencodes resistance to the herbicide chlorsulfuron.

Glyphosate resistance is imparted by mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and aroA genes. See, forexample, U.S. Pat. No. 4,940,835 to Shah et al., which discloses thenucleotide sequence of a form of EPSPS which can confer glyphosateresistance. U.S. Pat. No. 5,627,061 to Barry et al. also describes genesencoding EPSPS enzymes. See also U.S. Pat. Nos. 6,248,876; 6,040,497;5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642;4,940,835; 5,866,775; 6,225,114; 6,130,366; 5,310,667; 4,535,060;4,769,061; 5,633,448; 5,510,471; RE 36,449; RE 37,287; and 5,491,288;and international publications WO 97/04103; WO 97/04114; WO 00/66746; WO01/66704; WO 00/66747 and WO 00/66748, which are incorporated herein byreference for this purpose. Glyphosate resistance is also imparted toplants that express a gene that encodes a glyphosate oxido-reductaseenzyme as described more fully in U.S. Pat. Nos. 5,776,760 and5,463,175, which are incorporated herein by reference for this purpose.In addition glyphosate resistance can be imparted to plants by theover-expression of genes encoding glyphosate N-acetyltransferase. See,for example, U.S. patent application Ser. Nos. 10/004,357; 10/427,692,and 10/835,615.

Sterility genes can also be encoded in a DNA construct and provide analternative to physical detasseling. Examples of genes used in such waysinclude male tissue-preferred genes and genes with male sterilityphenotypes such as QM, described in U.S. Pat. No. 5,583,210. Other genesinclude kinases and those encoding compounds toxic to either male orfemale gametophytic development.

Commercial traits can also be encoded on a gene or genes that couldincrease for example, starch for ethanol production, or provideexpression of proteins. Another important commercial use of transformedplants is the production of polymers and bioplastics such as describedin U.S. Pat. No. 5,602,321. Genes such as β-Ketothiolase, PHBase(polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase (seeSchubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitateexpression of polyhyroxyalkanoates (PHAs).

Agronomically important traits that affect quality of grain, such aslevels and types of oils, saturated and unsaturated, quality andquantity of essential amino acids, levels of cellulose, starch, andprotein content can be genetically altered using the methods of theembodiments. Modifications include increasing content of oleic acid,saturated and unsaturated oils, increasing levels of lysine and sulfur,providing essential amino acids, and modifying starch. Hordothioninprotein modifications in corn are described in U.S. Pat. Nos. 5,990,389;5,885,801; 5,885,802 and 5,703,049; herein incorporated by reference.Another example is lysine and/or sulfur rich seed protein encoded by thesoybean 2S albumin described in U.S. Pat. No. 5,850,016, filed Mar. 20,1996, and the chymotrypsin inhibitor from barley, Williamson et al.(1987) Eur. J. Biochem. 165:99-106, the disclosures of which are hereinincorporated by reference.

Exogenous products include plant enzymes and products as well as thosefrom other sources including prokaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like.

Examples of other applicable genes and their associated phenotypeinclude the gene that encodes viral coat protein and/or RNA, or otherviral or plant genes that confer viral resistance; genes that conferfungal resistance; genes that confer insect resistance; genes thatpromote yield improvement; and genes that provide for resistance tostress, such as dehydration resulting from heat and salinity, toxicmetal or trace elements, or the like.

In other embodiments of the present invention, the ZmPOX24 promotersequences are operably linked to genes of interest that improve plantgrowth or increase crop yields under high plant density conditions. Forexample, the ZmPOX24 promoter may be operably linked to nucleotidesequences expressing agronomically important genes that result inimproved primary or lateral root systems. Such genes include, but arenot limited to, nutrient/water transporters and growth inducers.Examples of such genes, include but are not limited to, maize plasmamembrane H⁺-ATPase (MHA2) (Frias et al. (1996) Plant Cell 8:1533-44);AKT1, a component of the potassium uptake apparatus in Arabidopsis(Spalding et al. (1999) J. Gen. Physiol. 113:909-18); RML genes, whichactivate cell division cycle in the root apical cells (Cheng et al.(1995) Plant Physiol. 108:881); maize glutamine synthetase genes(Sukanya et al. (1994) Plant Mol. Biol. 26:1935-46); and hemoglobin(Duff et al. (1997) J. Biol. Chem. 27:16749-16752; Arredondo-Peter etal. (1997) Plant Physiol. 115:1259-1266; Arredondo-Peter et al. (1997)Plant Physiol. 114:493-500 and references cited therein). The ZmPOX24promoter may also be useful in expressing antisense nucleotide sequencesof genes that negatively affect root development under high-plantingdensity conditions.

“RNAi” refers to a series of related techniques to reduce the expressionof genes (See for example U.S. Pat. No. 6,506,559). Older techniquesreferred to by other names are now thought to rely on the samemechanism, but are given different names in the literature. Theseinclude “antisense inhibition,” the production of antisense RNAtranscripts capable of suppressing the expression of the target protein,and “co-suppression” or “sense-suppression,” which refer to theproduction of sense RNA transcripts capable of suppressing theexpression of identical or substantially similar foreign or endogenousgenes (U.S. Pat. No. 5,231,020, incorporated herein by reference). Suchtechniques rely on the use of constructs resulting in the accumulationof double stranded RNA with one strand complementary to the target geneto be silenced. The ZmPOX24 promoter sequence of the embodiments, andits related biologically active fragments or variants disclosed herein,may be used to drive expression of constructs that will result in RNAinterference including microRNAs and siRNAs.

The heterologous nucleotide sequence operably linked to the ZmPOX24promoter and its related biologically active fragments or variantsdisclosed herein may be an antisense sequence for a targeted gene. Theterminology “antisense DNA nucleotide sequence” is intended to mean asequence that is in inverse orientation to the 5′-to-3′ normalorientation of that nucleotide sequence. When delivered into a plantcell, expression of the antisense DNA sequence prevents normalexpression of the DNA nucleotide sequence for the targeted gene. Theantisense nucleotide sequence encodes an RNA transcript that iscomplementary to and capable of hybridizing to the endogenous messengerRNA (mRNA) produced by transcription of the DNA nucleotide sequence forthe targeted gene. In this case, production of the native proteinencoded by the targeted gene is inhibited to achieve a desiredphenotypic response. Modifications of the antisense sequences may bemade as long as the sequences hybridize to and interfere with expressionof the corresponding mRNA. In this manner, antisense constructionshaving 70%, 80%, 85% sequence identity to the corresponding antisensesequences may be used. Furthermore, portions of the antisensenucleotides may be used to disrupt the expression of the target gene.Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200nucleotides, or greater may be used. Thus, the promoter sequencesdisclosed herein may be operably linked to antisense DNA sequences toreduce or inhibit expression of a native protein in the plant.

In one embodiment of the invention, DNA constructs will comprise atranscriptional initiation region comprising one of the promoternucleotide sequences disclosed herein, or variants or fragments thereof,operably linked to a heterologous nucleotide sequence whose expressionis to be controlled by the inducible promoter of the embodiments. Such aDNA construct is provided with a plurality of restriction sites forinsertion of the nucleotide sequence to be under the transcriptionalregulation of the regulatory regions. The DNA construct may additionallycontain selectable marker genes.

The DNA construct will include in the 5′-3′ direction of transcription,a transcriptional initiation region (i.e., an inducible promoter of theembodiments), translational initiation region, a heterologous nucleotidesequence of interest, a translational termination region and,optionally, a transcriptional termination region functional in the hostorganism. The regulatory regions (i.e., promoters, transcriptionalregulatory regions, and translational termination regions) and/or thepolynucleotide of the embodiments may be native/analogous to the hostcell or to each other. Alternatively, the regulatory regions and/or thepolynucleotide of the embodiments may be heterologous to the host cellor to each other. As used herein, “heterologous” in reference to asequence is a sequence that originates from a foreign species, or, iffrom the same species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous polynucleotide isfrom a species different from the species from which the polynucleotidewas derived, or, if from the same/analogous species, one or both aresubstantially modified from their original form and/or genomic locus, orthe promoter is not the native promoter for the operably linkedpolynucleotide.

The optionally included termination region may be native with thetranscriptional initiation region, may be native with the operablylinked polynucleotide of interest, may be native with the plant host, ormay be derived from another source (i.e., foreign or heterologous) tothe promoter, the polynucleotide of interest, the host, or anycombination thereof. Convenient termination regions are available fromthe Ti-plasmid of A. tumefaciens, such as the octopine synthase andnopaline synthase termination regions. See also Guerineau et al. (1991)Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfaconet al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989)Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic AcidsRes. 15:9627-9639. In particular embodiments, the potato proteaseinhibitor 11 gene (PinII) terminator is used. See, for example, Keil etal. (1986) Nucl. Acids Res. 14:5641-5650; and An et al. (1989) PlantCell 1:115-122, herein incorporated by reference in their entirety.

The DNA construct comprising a promoter sequence of the embodimentsoperably linked to a heterologous nucleotide sequence may also containat least one additional nucleotide sequence for a gene to becotransformed into the organism. Alternatively, the additionalsequence(s) can be provided on another DNA construct.

Where appropriate, the heterologous nucleotide sequence whose expressionis to be under the control of the inducible promoter sequence of theembodiments and any additional nucleotide sequence(s) may be optimizedfor increased expression in the transformed plant. That is, thesenucleotide sequences can be synthesized using plant preferred codons forimproved expression. Methods are available in the art for synthesizingplant-preferred nucleotide sequences. See, for example, U.S. Pat. Nos.5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of theheterologous nucleotide sequence may be adjusted to levels average for agiven cellular host, as calculated by reference to known genes expressedin the host cell. When possible, the sequence is modified to avoidpredicted hairpin secondary mRNA structures.

The DNA constructs may additionally contain 5′ leader sequences. Suchleader sequences can act to enhance translation. Translation leaders areknown in the art and include: picornavirus leaders, for example, EMCVleader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al.(1989) Proc. Nat. Acad. Sci. USA 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Allison et al. (1986) Virology154:9-20); MDMV leader (Maize Dwarf Mosaic Virus); human immunoglobulinheavy-chain binding protein (BiP) (Macejak et al. (1991) Nature353:90-94); untranslated leader from the coat protein mRNA of alfalfamosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625);tobacco mosaic virus leader (TMV) (Gallie et al. (1989) MolecularBiology of RNA, pages 237-256); and maize chlorotic mottle virus leader(MCMV) (Lommel et al. (1991) Virology 81:382-385). See also Della-Cioppaet al. (1987) Plant Physiology 84:965-968. Other methods known toenhance translation and/or mRNA stability can also be utilized, forexample, introns, such as the maize Ubiquitin intron (Christensen andQuail (1996) Transgenic Res. 5:213-218; Christensen et al. (1992) PlantMolecular Biology 18:675-689) or the maize Adhl intron (Kyozuka et al.(1991) Mol. Gen. Genet. 228:40-48; Kyozuka et al. (1990) Maydica35:353-357), and the like.

The DNA constructs of the embodiments can also include furtherenhancers, either translation or transcription enhancers, as may berequired. These enhancer regions are well known to persons skilled inthe art, and can include the ATG initiation codon and adjacentsequences. The initiation codon must be in phase with the reading frameof the coding sequence to ensure translation of the entire sequence. Thetranslation control signals and initiation codons can be from a varietyof origins, both natural and synthetic. Translational initiation regionsmay be provided from the source of the transcriptional initiationregion, or from the structural gene. The sequence can also be derivedfrom the regulatory element selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. It isrecognized that to increase transcription levels enhancers may beutilized in combination with the promoter regions of the embodiments.Enhancers are known in the art and include the SV40 enhancer region, the35S enhancer element, and the like.

In preparing the DNA construct, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites. Restriction sites may be added or removed,superfluous DNA may be removed, or other modifications of the like maybe made to the sequences of the embodiments. For this purpose, in vitromutagenesis, primer repair, restriction, annealing, re-substitutions,for example, transitions and transversions, may be involved.

Reporter genes or selectable marker genes may be included in the DNAconstructs. Examples of suitable reporter genes known in the art can befound in, for example, Jefferson et al. (1991) in Plant MolecularBiology Manual, ed. Gelvin et al. (Kluwer Academic Publishers), pp.1-33; DeWet et al. (1987) Mol. Cell. Biol. 7:725-737; Goff et al. (1990)EMBO J. 9:2517-2522; Kain et al. (1995) BioTechniques 19:650-655; andChiu et al. (1996) Current Biology 6:325-330.

Selectable marker genes for selection of transformed cells or tissuescan include genes that confer antibiotic resistance or resistance toherbicides. Examples of suitable selectable marker genes include, butare not limited to, genes encoding resistance to chloramphenicol(Herrera Estrella et al. (1983) EMBO J. 2:987-992); methotrexate(Herrera Estrella et at (1983) Nature 303:209-213; Meijer et al. (1991)Plant Mol. Biol. 16:807-820); hygromycin (Waldron et al. (1985) PlantMol Biol. 5:103-108; Zhijian et al. (1995) Plant Science 108:219-227);streptomycin (Jones et al. (1987) Mol. Gen. Genet. 210:86-91);spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res.5:131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol. 7:171-176);sulfonamide (Guerineau et al. (1990) Plant Mol. Biol. 15:127-136);bromoxynil (Stalker et al. (1988) Science 242:419-423); glyphosate (Shawet al. (1986) Science 233:478-481); phosphinothricin (DeBlock et al.(1987) EMBO J. 6:2513-2518).

Other genes that could serve utility in the recovery of transgenicevents but might not be required in the final product would include, butare not limited to, examples such as GUS (b-glucuronidase; Jefferson(1987) Plant Mol. Biol. Rep. 5:387), GFP (green florescence protein;Chalfie et al. (1994) Science 263:802), luciferase (Riggs et al. (1987)Nucleic Acids Res. 15(19):8115 and Luehrsen et al. (1992) MethodsEnzymol. 216:397414), and the maize genes encoding for anthocyaninproduction (Ludwig et al. (1990) Science 247:449).

The nucleic acid molecules of the embodiments are useful in methodsdirected to expressing a nucleotide sequence in a plant. This may beaccomplished by transforming a plant cell of interest with a DNAconstruct comprising a promoter identified herein, operably linked to aheterologous nucleotide sequence, and regenerating a stably transformedplant from said plant cell. The methods of the embodiments are alsodirected to inducibly expressing a nucleotide sequence in a plant. Thosemethods comprise transforming a plant cell with a DNA constructcomprising a promoter identified herein that initiates transcription ina plant cell in an inducible manner, operably linked to a heterologousnucleotide sequence, regenerating a transformed plant from said plantcell, and subjecting the plant to the required stimulus to induceexpression.

The DNA construct comprising the particular promoter sequence of theembodiments operably linked to a nucleotide sequence of interest can beused to transform any plant. In this manner, genetically modified, i.e.transgenic or transformed, plants, plant cells, plant tissue, seed,root, and the like can be obtained.

Plant species suitable for the embodiments include, but are not limitedto, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),particularly those Brassica species useful as sources of seed oil,alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale),sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet(Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet(Setaria italica), finger millet (Eleusine coracana)), sunflower(Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticumaestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato(Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypiumbarbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya(Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera),pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobromacacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaamericana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifera indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardium occidentale), macadamia (Macadamia integrifolia),almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane(Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the embodiments include, forexample, pines such as loblolly pine (Pinus taeda), slash pine (Pinuselliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinuscontorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsugamenziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Piceaglauca); redwood (Sequoia sempervirens); true firs such as silver fir(Abies amabilis) and balsam fir (Abies balsamea); and cedars such asWestern red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparisnootkatensis). Plants of the embodiments may be crop plants (forexample, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower,peanut, sorghum, wheat, millet, tobacco, etc.). For example, thisinvention is suitable for any member of the monocot plant familyincluding, but not limited to, maize, rice, barley, oats, wheat,sorghum, rye, sugarcane, pineapple, yams, onion, banana, coconut, anddates.

As used herein, “vector” refers to a DNA molecule such as a plasmid,cosmid, or bacterial phage for introducing a nucleotide construct, forexample, a DNA construct, into a host cell. Cloning vectors typicallycontain one or a small number of restriction endonuclease recognitionsites at which foreign DNA sequences can be inserted in a determinablefashion without loss of essential biological function of the vector, aswell as a marker gene that is suitable for use in the identification andselection of cells transformed with the cloning vector. Marker genestypically include genes that provide tetracycline resistance, hygromycinresistance, or ampicillin resistance.

The methods of the embodiments involve introducing a nucleotideconstruct into a plant. As used herein “introducing” is intended to meanpresenting to the plant the nucleotide construct in such a manner thatthe construct gains access to the interior of a cell of the plant. Themethods of the embodiments do not depend on a particular method forintroducing a nucleotide construct to a plant, only that the nucleotideconstruct gains access to the interior of at least one cell of theplant. Methods for introducing nucleotide constructs into plants areknown in the art including, but not limited to, stable transformationmethods, transient transformation methods, and virus-mediated methods.

A “stable transformation” is one in which the nucleotide constructintroduced into a plant integrates into the genome of the plant and iscapable of being inherited by progeny thereof. “Transienttransformation” means that a nucleotide construct introduced into aplant does not integrate into the genome of the plant.

The nucleotide constructs of the embodiments may be introduced intoplants by contacting plants with a virus or viral nucleic acids.Generally, such methods involve incorporating a nucleotide construct ofthe embodiments within a viral DNA or RNA molecule. Methods forintroducing nucleotide constructs into plants and expressing a proteinencoded therein, involving viral DNA or RNA molecules, are known in theart. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785,5,589,367, and 5,316,931; herein incorporated by reference.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,981,840 and5,563,055), direct gene transfer (Paszkowski et al., (1984) EMBO J.3:2717-2722), and ballistic particle acceleration (see, for example,U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al.(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al.(1988) Biotechnology 6:923-926). Also see Weissinger et al. (1988) Ann.Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science andTechnology 5:27-37 (onion); Christou et al. (1988) Plant Physiol.87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926(soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990)Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl.Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al.(1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) PlantCell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having inducible expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that inducible expression of the desired phenotypiccharacteristic is stably maintained and inherited and then seedsharvested to ensure inducible expression of the desired phenotypiccharacteristic has been achieved. Thus as used herein, “transformedseeds” refers to seeds that contain the nucleotide construct stablyintegrated into the plant genome.

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated. The regeneration, development and cultivation of plantsfrom single plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, (1988) In.:Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc., SanDiego, Calif.). This regeneration and growth process typically includesthe steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of theembodiments containing a desired polypeptide is cultivated using methodswell known to one skilled in the art.

The embodiments provide compositions for screening compounds thatmodulate expression within plants. The vectors, cells, and plants can beused for screening candidate molecules for agonists and antagonists ofthe ZmPOX24 promoter. For example, a reporter gene can be operablylinked to a ZmPOX24 promoter and expressed as a transgene in a plant.Compounds to be tested are added and reporter gene expression ismeasured to determine the effect on promoter activity.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

The embodiments are further defined in the following Examples, in whichparts and percentages are by weight and degrees are Celsius, unlessotherwise stated. Techniques in molecular biology were typicallyperformed as described in Ausubel or Sambrook, supra. It should beunderstood that these Examples, while indicating embodiments of theinvention, are given by way of illustration only. From the abovediscussion and these Examples, one skilled in the art can ascertain theessential characteristics of the embodiments, and without departing fromthe spirit and scope thereof, can make various changes and modificationsof them to adapt to various usages and conditions. Thus, variousmodifications of the embodiments in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

Example 1 Expression Pattern of the ZmPOX24 Gene

Evidence that the ZmPOX24 is expressed in an inducible manner wasobtained using Massively Parallel Signature Sequencing technology (MPSS)(see Brenner S, et al. (2000) Nature Biotechnology 18:630-634, Brenner Set al. (2000) Proc Natl Acad Sci USA 97:1665-1670). This technologyinvolves the generation of 17 base signature tags from mRNA samples thathave been reverse transcribed. The tags are simultaneously sequenced andassigned to genes or ESTs. The abundance of these tags is given a numbervalue that is normalized to parts per million (PPM) which then allowsthe tag expression, or tag abundance, to be compared across differenttissues. Thus, the MPSS platform can be used to determine the expressionpattern of a particular gene and its expression level in differenttissues.

An EST sequence corresponding to the ZmPOX24 gene was entered into theMPSS database. The signature tag was identified and found to be highlyinduced in libraries of maize tissues infested with insects. Forexample, in the roots of maize plants infested with corn rootworm (CRW)for 24 hours, the abundance of the signature tag was approximately 4150ppm. A comparison to other uninfested root libraries in the MPSSdatabase indicated the peroxidase gene is induced approximately 50-fold(the average expression level was 81 ppm). Similarly, the signature tagwas induced in leaf/whorl tissue after a 24-hour infestation with ECBlarvae. The level of induction was approximately 180-fold greater thanin uninfested leaf libraries (avg. 10 ppm). No expression was indicatedin other vegetative tissue or in pollen. In kernels, expression wasindicated in only 1 library (endosperm-pericarp) at a level of 13 ppm.

The combination of the CRW and ECB induction of expression indicates theperoxidase promoter is a suitable candidate to direct significant levelsof transgene expression in plants, such as maize, in cases where insector wound induction of transgene expression is desired.

Example 2 Northern Analysis of ZmPOX24 Expression

Northern blot analysis was performed to further demonstrate theinducible expression of the ZmPOX24 gene. RNA derived from leaves androots of CRW-infested and uninfested V5 stage (5 collared leaves) B73maize plants was electrophoresed, blotted, and hybridized with probessynthesized from the ZmPOX24 EST.

Strong hybridization was only detected in RNA samples from root tissueof CRW-infested plants. Low levels of expression were detected in theroots of uninfested plants. No expression was detected in the leaves ofinfested or uninfested plants. These results corroborate the MPSSresults and provide further evidence that the ZmPOX24 gene is expressedat high levels in response to insect feeding.

Materials and Methods Utilized

Maize plants from the line, B73, were grown under greenhouse conditionsto the stage of V5 and infested with 75 2^(nd) instar WCRW. Leaves (V5leaf) and nodal roots were harvested. Uninfested V5 stage plants werealso sampled. RNA was extracted from the tissues using RNA Easy Maxi Kit(Qiagen, Valencia, Calif.)

The RNA samples were electrophoresed through a standard formaldehyde gelusing Northern Max Denaturing Gel Buffers (Ambion, Austin, Tex.)Afterwards, the gel was washed once in 20×SSC for a total of 15 minutes,then blotted to a nylon membrane overnight in 20×SSC [Turboblotter fromS&S.] The membrane was UV-crosslinked for 2 minutes. Pre-hybridizationof the blot totaled 3 hours at 50° C. in 15 mL DIG Easy Hyb Solution(Roche Applied Sciences, Indianapolis, Ind.) Single-stranded DNA probeswere made from the ZmPOX24 EST by PCR using the PCR DIG Probe SynthesisKit (Roche). Following the kit protocol recommendations, 2 μL ofdenatured probe was added per 1 mL of DIG Easy Hyb solution.Hybridization was allowed to go overnight at 50° C. in 10 mL ofsolution. The next day the membrane was washed twice in Low StringencyBuffer (2×SSC containing 0.1% SDS) at room temperature for 5 minutes,and then washed twice in High Stringency Buffer (0.1×SSC containing 0.1%SDS) at 55° C. for 15 minutes.

Visualization of the ZmPOX24 transcripts was accomplished using the DIGOMNI System for PCR Probes and DIG Wash and Block Buffer Set from Roche.Briefly, the membrane was equilibrated in Washing Buffer (0.1 M MaleicAcid, 0.15 M NaCl, pH 7.5; 0.3% (v\v) Tween 20) for 2 minutes at roomtemperature, then put in Blocking Solution (1:10 dilution of 10×Blocking Solution in Maleic Acid Buffer (0.1 M Maleic Acid, 0.15 M NaCl,pH 7.5)) for 30 minutes. The membrane was incubated in Antibody Solution(anti-DIG-alkaline phosphate diluted 1:10,000 in Blocking Solution (10×Blocking Solution diluted 1:10 in Maleic Acid Buffer)) for 30 minutesand washed 2 times in Washing Buffer for a total of 30 minutes. Afterequilibration in Detection Buffer (0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5)for 3 minutes, the membrane was placed in a plastic development folder(Applied BioSystems) and 1 mL of CDP-Star Working Solution (1:100dilution of CDP-Star (25 mM, 12.38 mg/ml) in Detection Buffer) wasapplied drop-wise to the membrane. Following a 5 minute incubation atroom temperature, the membrane was exposed to X-ray film from 20 minutesto overnight.

Example 3 Isolation of the Promoter for the ZmPOX24 Gene, and SequenceAnalysis

Analysis of the ZmPOX24 gene indicated it was expressed at high levelsupon insect feeding. Thus, the ZmPOX24 promoter could be used to directhigh levels of expression in maize plants subject to insect pestinfestation.

A promoter of approximately 996 bp was isolated from the peroxidasegene. The sequence was obtained using multiple reiterative BLASTNsearches of the Genome Survey Sequence (GSS) database, starting withsequence from the EST, P0019.CLWAG71:FIS.

Each search of the GSS database used available sequence obtained fromthe previous search to “walk” along genomic sequence until no furtheroverlapping sequence was identified.

Analysis of the promoter sequence obtained indicated the presence ofsome interesting motifs. The ATATT motif, previously identified as beingpresent in other promoters with root specific expression (Elmayan &Tepfer (1995) Transgenic Research 4, 388-396), was identified in theZmPOX24 promoter in three separate locations. These can be seen in FIG.2.

The TATA box was identified and is indicated in FIG. 2. It is located atpositions 858 through 865 of SEQ ID NO: 1.

Two CNGTTR motifs have been identified in the sequence and are shown inFIG. 2. All animal MYB proteins and at least two plant MYB proteinsATMYB1 and ATMYB2, both isolated from Arabidopsis, bind to sequencescontaining the core CNGTT(A/G) element. ATMYB2 is involved in regulationof genes that are responsive to water stress in Arabidopsis. A petuniaMYB protein (MYB.Ph3) is involved in regulation of flavonoidbiosynthesis.

An AACGTGT motif has also been identified in the ZmPOX24 promotersequence and is shown in FIG. 2. This motif is a G-box-like motif andmay play a role in regulating quantitative expression (Elliot & Shirsat(1998) Plant Mol Biol 37: 675-687).

Example 4 Promoter Activity of ZmPOX24

To demonstrate that the DNA isolated as the ZmPOX24 promoter functionsas a promoter, transient particle bombardment assays were performed tolook for expression of the GUS reporter gene. These assays provide arapid assessment of whether a DNA fragment is able to direct geneexpression.

A 996 bp fragment was PCR amplified from genomic DNA and cloned into anexpression vector in front of the B-glucuronidase (GUS) gene to testwhether the DNA would direct expression. Test constructs of ZmPOX24 withand without the Adh1 intron were evaluated to look for any contributionof the intron to increased GUS expression. Numerous GUS staining foci onthe coleoptile (>30 foci/coleoptile) of seedlings were observed afterbiolistic bombardment of 3-day-old maize seedlings. The level ofstaining was less than a control which consisted of the strong,constitutive promoter, Ubi-1, directing GUS expression. This resultsupports the function of the 996 bp ZmPOX24 DNA as a promoter intransiently transformed maize cells.

Materials and Methods Utilized for the Biolistic Transient RootExpression Assay

B73 seeds were placed along one edge of germination paper soaked in asolution of 7% sucrose. An additional piece of germination paper,identical in size to the first, was also soaked in 7% sucrose and wasused to overlay the kernels. The germination paper—kernel—germinationpaper sandwich was subsequently rolled and placed into a beaker of 7%sucrose solution, such that the solution would wick up the paper to thekernels at the top of the roll. This allowed for straight root growth.Kernels were permitted to germinate and develop for 2-3 days in the darkat 27-28° C. prior to bombardment: The coleoptile was removed and theseedlings were placed in a sterile petri dish (60 mm) on a layer filterpaper moistened with distilled water. Two seedlings per plate werearranged in opposite orientations and anchored to the filter paper witha 0.5% agarose solution. The outer sheath covering the coleoptile wasremoved and seedlings were placed in a sterile petri dish (60 mm) on alayer of Whatman #1 filter paper moistened with 1 mL of H₂O. Twoseedlings per plate were arranged in opposite orientations and anchoredto the filter paper with a 0.5% agarose solution.

DNA/gold particle mixtures were prepared for bombardment in thefollowing method. Sixty mg of 0.6-1.0 micron gold particles werepre-washed with ethanol, rinsed with sterile distilled H₂O, andresuspended in a total of 1 mL of sterile H₂O. 50 μL aliquots of goldparticle suspension were stored in siliconized Eppendorf tubes at roomtemperature. DNA was precipitated onto the surface of the gold particlesby combining, in the following order, 50 μL aliquot of pre-washed 0.6 μMgold particles, 5-10 μg of test DNA, 50 μL 2.5 M CaCl₂ and 25 μL of 0.1M spermidine. The solution was immediately vortexed for 3 minutes andcentrifuged briefly to pellet the DNA/gold particles. The DNA/gold waswashed once with 500 μL of 100% ethanol and suspended in a final volumeof 50 μL of 100% ethanol. The DNA/gold solution was incubated at −20° C.for at least 60 minutes prior to applying 6 μL of the DNA/gold mixtureonto each Mylar macrocarrier.

Seedlings prepared as indicated above were bombarded with DNA/goldparticles twice using the PDS-1000/He gun at 1100 psi under 27-28 inchesof Hg vacuum. The distance between macrocarrier and stopping screen wasbetween 6-8 cm. Plates were incubated in sealed containers for 18-24 hin the dark at 27-28° C. following bombardment.

The bombarded seedlings, root tips, and leaf sections were assayed fortransient GUS expression by immersing in 10-15 mL of GUS assay buffercontaining 100 mM NaH₂PO₄—H₂O (pH 7.0), 10 mM EDTA, 0.5 mMK₄Fe(CN)₆-3H₂O, 0.1% Triton X-100 and 2 mM 5-bromo4-chloro-3-indoylglucuronide. The tissues were incubated in the dark for 24 hours at 37°C. Replacing the GUS staining solution with 100% ethanol stopped theassay. GUS expression/staining was visualized under a microscope.

Example 5 Expression of ZmPOX24 in Transgenic Plants

Stable transformed plants were created using Agrobacteriumtransformation protocols as per Example 6 to allow for a more detailedcharacterization of promoter activity, including induction of thepromoter via mechanical wounding and wounding via insect feeding.

Leaf and root tissue from regenerated plants growing on nutrient agarthat are stably transformed with DNA constructs containing the 996 bpZmPOX24 promoter (SEQ ID NO:1) operably connected to the GUS gene(abbreviated as ZmPOX24:GUS) or the 996 bp ZmPOX24 promoter operablylinked to the Adh1 intron and the GUS gene (abbreviated as ZmPOX24(Adh1intron1):GUS) were sampled to test for GUS enzyme activity. The Adh1intron was included for the purpose of increasing expression as it hasbeen shown in cereal cells that the expression of foreign genes isenhanced by the presence of an intron in gene constructs (See Callis etal. (1987) Genes and Development 1: 1183-1200; Kyozuka et al. (1990)Maydica 35:353-357). Histochemical analysis showed GUS was expressed inthe leaves of approximately half of the events, independent of thepresence or absence of the Adh1 intron. In contrast, a 2-fold increasein the number of GUS expressing events (35% vs 18%) was observed in roottissue with the Adh1 intron. The inducibility of the ZmPOX24 promoterwas tested at the plantlet stage by crushing the first 0.75 cm of theroot tip with serrated forceps and harvesting the wounded root and aleaf 24 hours after wounding. No GUS induction above that observed inunwounded plantlet controls was detected in leaves after root woundingwith or without the Adh1 intron. In roots, however, the number of eventsexpressing GUS increased to approximately 68%. Much of the expressionthat was observed was located adjacent to the wounded tissue. Asignificant increase in the percentage of events expressing GUS in rootswas not observed with ADH intron events. These plantlet results reflectthe Lynx MPSS results in Example 1 which indicated that the ZmPOX24promoter is inducible.

Additional characterization of the ZmPOX24 promoter was performed ontransgenic plants in the greenhouse. GUS expression was evaluated undernormal growing conditions, under stress by wounding, and under CRWfeeding pressure to distinguish whether there was a differentialresponse of ZmPOX24 to wounding and insect feeding. The assessment undernormal greenhouse conditions provided a baseline by which to assessinduction in the plants, Nodal roots and the V5 leaf of V5 staged (5collared leaves) ZmPOX24 plants were simultaneously wounded by crushingabout 1 inch of the root tip with serrated pliers and by removing theabaxial 1-2 inches of the leaf. Twenty-four hours later, the plants weresampled. Results showed ZmPOX24 was up regulated in each event examined.The induction occurred not only at the wound site, but also in unwoundedportions of each wounded organ.

In contrast to wounding, the induction of the ZmPOX24 promoter was morelocalized in response to CRW feeding. Induction with CRW was performedby allowing CRW larvae to feed on the roots for 48 hours. At 48 hoursthe injured roots and leaves from these plants were subjected tohistochemical staining to identify where induction occurred. The resultsshowed an increase in the level of GUS expression in the roots which waslocalized to the area at or near the CRW feeding site.

The results of quantitative fluorometric assays on leaf and root tissuefrom ZmPOX24 events that were mechanically wounded at the root or by CRWfeeding is presented in Table 1. They reflect the observations made withthe histochemical analysis and further show differences in inductionbetween mechanically wounded and CRW-injured roots. The level of GUSexpression was between 2.5 and 3.5-fold higher in CRW injured root tipscompared to wounded root tips. This was expected, based on the morelocalized induction with CRW feeding than with wounding. Smallerdifferences in the mature region of the root were observed between thetwo treatments. In both cases a preference for root expression wasobserved when expression levels in roots were compared with leaf tissue.These results suggest that induction of expression directed by theZmPox24 promoter by CRW feeding is largely limited to the root. It alsodemonstrates that the ZmPOX24 promoter responds differently tomechanical wounding and insect feeding.

TABLE 1 MUG Assay Results For ZmPOX24 (SEQ ID NO:1) Normalized ResultsLeaf Root Tip Mature Region Peroxidase (wounded) 0 17 12 Peroxidase: ADH(wounded) 13 40 45 Peroxidase (CRW) 0 41 16 Peroxidase: ADH (CRW) 16 14673 untransformed 0 0 0 (negative control) Values provided are medianvalues, as nmole MU/mg total protein/hr MU = 4-methyl umbelliferone MUG= 4-methyl umbelliferyl-B-D-glucuronide

Additional tissues such as silks, tassels, pollen, and kernels weresampled for GUS expression when plants reached the R1 and R2developmental stages (pollen shed and silking) under normal greenhousegrowing conditions. In silks, most of the plants showed some type ofexpression in approximately 10% of the silk strands in the samplepopulation. The percentage of silk strands expressing GUS increased to20% or greater when ZmPOX24 was in combination with the Adh1 intron. Fortassels, the majority of the ZmPOX24 promoter events showed weak to veryweak staining. However, many of the ADH1 intron containing eventsdemonstrated good expression in tassels. No GUS expression was observedin pollen in any of the events.

The ZmPOX24 promoter was active in the kernels most of the eventsanalyzed. Nearly all of this expression was identified in the embryo andnot in other tissues, such as the pericarp, aleurone, endosperm, orscutellum. Most events had expression in the coleoptile and/or plumule,followed by the 1^(st) internode. Only two events had expression in theprimary root.

Histochemical Staining of Plant Tissues for GUS Activity

Detection of GUS activity was accomplished by placing tissue fromtransformed plants into 48-well, 12-well or 6-well plates containing 0.5to 5 mL GUS assay buffer (assay buffer recipe described in Example 4).Plates were placed under house vacuum for 10 min, then incubatedovernight at 37° C. Tissue was cleared of pigmentation with 1 to 3successive 12 hour incubations in 100% ethanol at room temperature. Thetissues were stored in 70% ethanol at 4° C.

Example 6 Agrobacterium-Mediated Transformation of Maize andRegeneration of Transgenic Plants

For Agrobacterium-mediated transformation of maize with a promotersequence of the embodiments, the method of Zhao was employed (U.S. Pat.No. 5,981,840, (hereinafter the '840 patent) and PCT patent publicationWO98/32326; the contents of which are hereby incorporated by reference).

Agrobacterium were grown on a master plate of 800 medium and cultured at28° C. in the dark for 3 days, and thereafter stored at 4° C. for up toone month. Working plates of Agrobacterium were grown on 810 mediumplates and incubated in the dark at 28° C. for one to two days.

Briefly, embryos were dissected from fresh, sterilized corn ears andkept in 561Q medium until all required embryos were collected. Embryoswere then contacted with an Agrobacterium suspension prepared from theworking plate, in which the Agrobacterium contained a plasmid comprisingthe promoter sequence of the embodiments. The embryos were co-cultivatedwith the Agrobacterium on 562P plates, with the embryos placed axis downon the plates, as per the '840 patent protocol.

After one week on 562P medium, the embryos were transferred to 5630medium. The embryos were subcultured on fresh 5630 medium at 2 weekintervals and incubation was continued under the same conditions. Callusevents began to appear after 6 to 8 weeks on selection.

After the calli had reached the appropriate size, the calli werecultured on regeneration (288W) medium and kept in the dark for 2-3weeks to initiate plant regeneration. Following somatic embryomaturation, well-developed somatic embryos were transferred to mediumfor germination (272V) and transferred to a lighted culture room.Approximately 7-10 days later, developing plantlets were transferred to272V hormone-free medium in tubes for 7-10 days until plantlets werewell established. Plants were then transferred to inserts in flats(equivalent to 2.5″ pot) containing potting soil and grown for 1 week ina growth chamber, subsequently grown an additional 1-2 weeks in thegreenhouse, then transferred to classic 600 pots (1.6 gallon) and grownto maturity.

Media Used in Agrobacterium-Mediated Transformation and Regeneration ofTransgenic Maize Plants:

561Q medium comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 mL/LEriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/L thiamine HCl, 68.5g/L sucrose, 36.0 g/L glucose, 1.5 mg/L 2,4-D, and 0.69 g/L L-proline(brought to volume with dl H₂O following adjustment to pH 5.2 with KOH);2.0 g/L Gelrite™ (added after bringing to volume with dl H₂O); and 8.5mg/L silver nitrate (added after sterilizing the medium and cooling toroom temperature).

800 medium comprises 50.0 mL/L stock solution A and 850 mL dl H₂O, andbrought to volume minus 100 mL/L with dl H₂O, after which is added 9.0 gof phytagar. After sterilizing and cooling, 50.0 mL/L stock solution Bis added, along with 5.0 g of glucose and 2.0 mL of a 50 mg/mL stocksolution of spectinomycin. Stock solution A comprises 60.0 g of dibasicK₂HPO₄ and 20.0 g of monobasic sodium phosphate, dissolved in 950 mL ofwater, adjusted to pH 7.0 with KOH, and brought to 1.0 L volume with dlH₂O, Stock solution B comprises 20.0 g NH₄Cl, 6.0 g MgSO₄.7H₂O, 3.0 gpotassium chloride, 0.2 g CaCl₂, and 0.05 g of FeSO₄.7H₂O, all broughtto volume with dl H₂O, sterilized, and cooled.

810 medium comprises 5.0 g yeast extract (Difco), 10.0 g peptone(Difco), 5.0 g NaCl, dissolved in dl H₂O, and brought to volume afteradjusting pH to 6.8. 15.0 g of bacto-agar is then added, the solution issterilized and cooled, and 1.0 mL of a 50 mg/mL stock solution ofspectinomycin is added.

562P medium comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 mL/LEriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0g/L sucrose, and 2.0 mg/L 2,4-D (brought to volume with dl H₂O followingadjustment to pH 5.8 with KOH); 3.0 g/L Gelrite™ (added after bringingto volume with dl H₂O); and 0.85 mg/L silver nitrate and 1.0 mL of a 100mM stock of acetosyringone (both added after sterilizing the medium andcooling to room temperature).

5630 medium comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 mL/LEriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0g/L sucrose, 1.5 mg/L 2,4-D, 0.69 g L-proline, and 0.5 g MES buffer(brought to volume with dl H₂O following adjustment to pH 5.8 with KOH).Then, 6.0 g/L Ultrapure™ agar-agar (EM Science) is added and the mediumis sterilized and cooled. Subsequently, 0.85 mg/L silver nitrate, 3.0 mLof a 1 mg/mL stock of Bialaphos, and 2.0 mL of a 50 mg/mL stock ofcarbenicillin are added.

288 W comprises 4.3 g/L MS salts (GIBCO 11117-074), 5.0 mL/L MS vitaminsstock solution (0.100 g nicotinic acid, 0.02 g/L thiamine HCl, 0.10 g/Lpyridoxine HCl, and 0.40 g/L Glycine brought to volume with polished D-lH₂O) (Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/Lmyo-inositol, 0.5 mg/L zeatin, and 60 g/L sucrose, which is then broughtto volume with polished D-l H₂O after adjusting to pH 5.6. Following,6.0 g/L of Ultrapure™ agar-agar (EM Science) is added and the medium issterilized and cooled. Subsequently, 1.0 mL/L of 0.1 mM abscisic acid;1.0 mg/L indoleacetic acid and 3.0 mg/L Bialaphos are added, along with2.0 mL of a 50 mg/mL stock of carbenicillin.

Hormone-free medium (272V) comprises 4.3 g/L MS salts (GIBCO 11117-074),5.0 mL/L MS vitamins stock solution (0.100 g/L nicotinic acid, 0.02 g/Lthiamine HCl, 0.10 g/L pyridoxine HCl, and 0.40 g/L Glycine brought tovolume with polished dl H₂O), 0.1 g/L myo-inositol, and 40.0 g/L sucrose(brought to volume with polished dl H₂O after adjusting pH to 5.6); and6 g/L Bacto-agar (added after bringing to volume with polished dl H₂O),sterilized and cooled to 60° C.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A DNA construct comprising an isolated nucleic acid moleculecomprising a nucleotide sequence that initiates transcription in a plantcell selected from the group consisting of: a) a nucleotide sequencecomprising SEQ ID NO: 1; b) a nucleotide sequence comprising the plantpromoter sequence of the plasmid deposited as Patent Deposit No.PTA-6276; and c) a nucleotide sequence comprising a fragment of thesequence set forth in SEQ ID NO: 1, wherein said fragment initiatestranscription in the plant cell in response to wounding or insectfeeding, wherein said isolated nucleic acid molecule is operably linkedto a heterologous nucleotide sequence of interest.
 2. A vectorcomprising the DNA construct of claim 1.